thapsigargin and Lymphoma--B-Cell

The phenotypically immature B cell lymphoma WEHI-231 undergoes apoptotic cell death when cultured with anti-immunoglobulin (Ig) antibodies, via a bcl-2-independent mechanism. We have therefore studied the role of the bcl-2-related protein bcl-x in controlling cell death in WEHI-231. We find that overexpression of the long form of bcl-x (bcl-XL) renders these cells refractory to anti-Ig-induced cell death. Stimulation of WEHI-231 via CD40 has similar protective effects. We show here that ligation of CD40 rapidly induces the appearance of the bcl-XL protein in WEHI-231, while stimulation via sIgM, sIgD, CD5 or CD45 receptors, or with phorbol esters plus ionomycin does not. WEHI-231 cells also rapidly undergo massive apoptosis following culture with thapsigargin, a specific inhibitor of the Ca(2+)-ATPase of the endoplasmic reticulum: this is also reversed by anti-CD40, or by overexpression of bcl-XL. We, therefore, conclude that bcl-XL plays a key role in the regulation of antigen receptor-mediated apoptosis via CD40 in WEHI-231. In addition, the fact that this protein is not induced in WEHI-231 in response to phorbol dibutyrate plus ionomycin points to a fundamental signaling defect in these cells, which could conceivably be a reflection of their immature, apoptosis-susceptible phenotype.

Topics: Animals; Antibodies, Anti-Idiotypic; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; Apoptosis; bcl-X Protein; CD40 Antigens; CD40 Ligand; Gene Expression Regulation; Humans; Ionomycin; Lymphoma, B-Cell; Membrane Glycoproteins; Mice; Phorbol 12,13-Dibutyrate; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Terpenes; Thapsigargin; Transfection; Tumor Cells, Cultured

The engagement of the B cell antigen receptor is the first step of antigenic stimulation of B lymphocytes. This step is followed by a series of biochemical events, including the activation of protein-tyrosine kinases, phosphoinositide turnover, and multiple patterns of calcium mobilization, which lead to the regulation of gene transcription and cellular responses. The B cell antigen receptor complex is composed of membrane immunoglobulins (as antigen recognition subunits) and associated chains (Ig-alpha and Ig-beta) that couple the receptor to cytoplasmic protein kinases. To investigate independently the relative signaling capacity of Ig-alpha and Ig-beta, chimeric proteins containing their cytoplasmic domains were expressed in a B cell line. We found that Ig-alpha and Ig-beta activate two distinct intracellular signaling pathways. The engagement of Ig-alpha chimeras induces a complete release of calcium from intracellular stores, followed by transmembrane calcium influx and late cell activation signals, detected by lymphokine secretion. In contrast, Ig-beta chimeras do not induce lymphokine secretion or calcium influx, but induce short oscillatory release of calcium, dependent on the activity of the Ca-ATPase pump of the endoplasmic reticulum. These results provide a structural basis for the diversity of B cell responses.

Topics: B-Lymphocytes; Calcium; Calcium Channels; Calcium-Transporting ATPases; Cell Line; Humans; Interleukin-2; Lymphoma, B-Cell; Macromolecular Substances; Models, Biological; Phosphoproteins; Phosphotyrosine; Receptors, Antigen, B-Cell; Recombinant Fusion Proteins; Signal Transduction; Terpenes; Thapsigargin; Transfection; Tumor Cells, Cultured; Type C Phospholipases; Tyrosine